Polysialic acids are synthesized by pathogenic bacteria as capsular polysaccharides. These polymers have been implicated in the virulence of some strains of Escherichia coli, which cause neonatal meningitis and urinary tract infections. Polysialic acid is also present in the developing human brain. There has been significant progress in identifying the gene necessary for capsular polysaccharide biosynthesis in gram negative bacteria. The objective of this project is to determine the mechanism of capsular polysaccharide biosynthesis in virulent encapulated bacteria. Our approach is to characterize the structure and function of the enzymes in the pathway and to use them as tools for understanding sialylation in bacteria and humans. Much of the enzymology of polysialic acid capsular polysaccharide synthesis has been done with the a(2-8) polysialyltransferase complex of E. coli K1. Bacteria containing DNA fragments encoding several capsule related genes have been used as a source of enzyme activity. As a model system for investigating the mechanism of capsular glycosyltransferases we have chosen to investigate the K92 a(2-8)(2-9) polysialyltransferase in a genetic background lacking other capsule related genes. We have cloned the gene for the K92 PST into an expression vector and solubliized the membrane bound enzyme with detergents. The enzyme require association with an intact membrane for activity. Purification of this protein is underway. The K92 PST requires an exogenously added acceptor when assayed in this genetic background. The K92 polysialyltranseferse does not require neuE gene product for activity. The effect of acceptor repeat unit structure, chain length on elongation activity was determined by the addition of other sialic acid polymers, oligosialic acid, and gangliosides. We developed a means to produce adequate quantities of K92 oligosaccharide. We isolated oligos ranging in size from dimer to 16mer. These oligos terminated predominantly in a a2,9 linkage. From these studies we have been able to determine a binding specificity for the exogenous acceptor. We have demonstrated a clear preference for acceptors that terminate in an a(2,8) linkage at the non-reducing end.